Fungus-farming termites (Macrotermitinae) engage in an obligate mutualism with members of the fungal genus Termitomyces, which they maintain as a monoculture on specialized comb structures. Both these comb structures and the guts of the termites host diverse bacterial communities that are believed to assist in sustaining monoculture farming through antagonist suppression. Among candidate bacteria-derived compounds serving this function are non-ribosomal peptides (NRPs), which are a highly bioactive class of specialized metabolites, frequently produced by symbionts within eukaryotic hosts. However, our understanding of specialized metabolites in termite-associated microbiomes is limited. Here we use amplicon sequencing to characterize both bacterial composition and NRP potential. We show that bacterial and NRP diversity are correlated and that the former varies more than the latter across termite host and gut and comb samples. Compositions of the two are governed by host species and sample type, with topological similarity indicating a diverse set of biosynthetic potential that is consistent with the long evolutionary history of the Macrotermitinae. The structure of both bacterial and NRP compositional networks varied similarly between guts and combs across the Macrotermitinae albeit with auxiliary termite genus-specific patterns. We observed minimal termite species-specific cores, with essentially no Macrotermitinae-wide core and an abundance of putatively novel biosynthetic gene clusters, suggesting that there is likely no single solution to antagonist suppression via specialized NRP metabolites. Our findings contribute to an improved understanding of the distribution of NRP potential in the farming termite symbiosis and will help guide targeted exploration of specialized metabolite production.

Uropygial gland secretions of birds consist of host and bacteria derived compounds and play a major sanitary and feather-protective role. Here we report on our microbiome studies of the New Guinean toxic bird Pachycephala schlegelii and the isolation of a member of the Amycolatopsis genus from the uropygial gland secretions. Bioactivity studies in combination with co-cultures, MALDI imaging and HR-MS/MS-based network analyses unveil the basis of its activity against keratinolytic bacteria and fungal skin pathogens. We trace the protective antimicrobial activity of Amycolatopsis sp. PS_44_ISF1 to the production of rifamycin congeners, ciromicin A and of two yet unreported compound families. We perform NMR and HR-MS/MS studies to determine the relative structures of six members belonging to a yet unreported lipopeptide family of pachycephalamides and of one representative of the demiguisins, a new hexapeptide family. We then use a combination of phylogenomic, transcriptomic and knock-out studies to identify the underlying biosynthetic gene clusters responsible for the production of pachycephalamides and demiguisins. Our metabolomics data allow us to map molecular ion features of the identified metabolites in extracts of P. schlegelii feathers, verifying their presence in the ecological setting where they exert their presumed active role for hosts. Our study shows that members of the Actinomycetota may play a role in avian feather protection.

The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a central role in the symbiosis through plant biomass decomposition and as the main food source for these termites. Several specialised (secondary) metabolites have been isolated from different Termitomyces species, suggesting that they may also aid in antimicrobial defence. Yet, we have a fragmented understanding of Termitomyces’ natural product repertoire. To determine the biochemical potential encoded by diverse Termitomyces species, we comparatively analysed 22 published and 17 newly generated genomes, spanning 21 of 52 described Termitomyces species and five of the 11 termite host genera. After extensive assembly and annotation optimisation, we employed fungiSMASH to detect 754 biosynthetic gene clusters (BGCs) coding for specialised metabolites. BiG-SCAPE analysis and manual curation allowed us to assign 660 of these BGCs to 61 distinct biosynthetic gene cluster families (GCFs), spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species, 21 GCFs were present in all genomes of several subsets of species, while the remaining 33 GCFs were inconsistently distributed across species. The 25 most abundant GCFs were subjected to codon-based evolutionary constraint analyses to evaluate their evolutionary histories and revealed two GCFs with consistent positive selection in the same gene across the phylogeny and seventeen genes with Termitomyces species-specific episodic positive selection. These patterns of selection indicate that millions of years of termite-fungus symbiosis have led to distinct evolutionary trajectories of biosynthetic gene clusters, ample putative chemical novelties, and uncover a vast non-random and largely unknown chemical potential of Termitomyces.

Motivation As previously described, amplicon analysis of the bacterial 16S gene has several limitations owing to fundamental characteristics of both the 16S gene and technological restrictions. Previously, RibDif was introduced to help quantify these limitations by detailed analysis of a given genera and the 16S gene profile of its members, notably multiplicity and divergence of 16S alleles within genomes as well as shared alleles between species. Apart from using amplicon analysis for only the 16S gene, amplicons derived from genus-specific genes or even functional genes are increasingly being utilized. Moreover, long-read technologies are progressively being used to sequence longer amplicons, and since these inherently contain more information, they may likely alleviate the issues proposed in RibDif. Results Taking these phenomena into account, we here propose RibDif2. RibDif2 retains the 16S-optimized functionality of the original RibDif but can now run any set of primers on any part of the genome in any set of organisms, be it prokaryote, eukaryote, or archaea. We demonstrate this new functionality by showing full species resolution of Pseudoalteromonas using complete rRNA-operon amplicons, as well as selection of optimally discriminatory primers for Staphylococcus and Pseudomonas. Moreover, we show a potential bias toward terrestrial bacteria relative to marine ones for primers amplifying biosynthetic gene clusters and lastly suggest optimal primers to differentiate the members of the insect genus Drosophila. We believe that RibDif2 will facilitate the work of all scientists using amplicon sequencing, especially in the era of long-read sequencing. Availability and implementation Ribdif2 is freely available at https://github.com/Rob-murphys/ribdif.

The Lactobacillaceae are lactic acid bacteria harnessed to deliver important outcomes across numerous industries, and their unambiguous, species-level identification from mixed community environments is an important endeavor. Amplicon-based metataxonomics using short-read sequencing of partial 16S rRNA gene regions is widely used to support this, however, the high genetic similarity among Lactobacillaceae species restricts our ability to confidently describe these communities even at genus level. Long-read sequencing (LRS) of the whole 16S rRNA gene or the near complete rRNA operon (16S-ITS-23S) has the potential to improve this. We explored species ambiguity amongst Lactobacillaceae using in-silico tool RibDif2, which identified allele overlap when various partial and complete 16S rRNA gene and 16S-ITS-23S rRNA regions were amplified. We subsequently implemented LRS by MinION™ to compare the capacity of V3–V4, 16S and 16S-ITS-23S rRNA amplicons to accurately describe the diversity of a 20-species Lactobacillaceae mock community in practice. In-silico analysis identified more instances of allele/species overlap with V3–V4 amplicons (n = 43) compared to the 16S rRNA gene (n = 11) and partial (n = up to 15) or complete (n = 0) 16S-ITS-23S rRNA amplicons. With subsequent LRS of a DNA mock community, 80% of target species were identified using V3–V4 amplicons whilst the 16S rRNA gene and 16S-ITS-23S rRNA region amplicons resulted in 95 and 100% of target species being identified. A considerable reduction in false-positive identifications was also seen with 16S rRNA gene (n = 3) and 16S-ITS-23S rRNA region (n = 9) amplicons compared with V3–V4 amplicons (n = 43). Whilst the target species affected by allele overlap in V3–V4 and 16S rRNA gene sequenced mock communities were predicted by RibDif2, unpredicted species ambiguity was observed in 16S-ITS-23S rRNA sequenced communities. Considering the average nucleotide identity (ANI) between ambiguous species (~97%) and the basecall accuracy of our MinION™ sequencing protocol (96.4%), the misassignment of reads between closely related taxa is to be expected. With basecall accuracy exceeding 99% for recent MinION™ releases, the increased species-level differentiating power promised by longer amplicons like the 16S-ITS-23S rRNA region, may soon be fully realized.

Fungus-farming termite colonies maintain monoculture fungus combs in underground chambers without apparent problems with diseases. Multiple lines of defense contribute to the suppression or removal of antagonists of the symbiosis, but the role of the termite-manipulated environment within mounds has yet to be tested. Specifically, termite mounds have extremely high levels of CO2 compared to atmospheric levels. We tested the effect of 5% CO2 on the growth of fungal crops from Macrotermes bellicosus colonies, generalist fungi that could challenge the symbiosis, as well as a specialist stowaway fungus, Pseudoxylaria. For sporulating fungi, we also quantified the effects on conidia production. We found that elevated CO2 significantly reduces mycelial growth and conidia production of the generalist fungi Aspergillus sp., Beauveria bassiana, and Metarhizium brunneum, whereas it overall had a net positive effect on the growth of the fungal crop Termitomyces and Pseudoxylaria; albeit, with variation between fungal strains within genera. Our findings point to elevated CO2 being of adaptive significance to the fungus-farming termite symbiosis as an additional layer of defense that helps keep termite fungus gardens free from fungal infections. The mound-building activities that make termites ecosystem engineers may thus also generate environmental conditions that impact the fate of fungi inhabiting the extended phenotypes that massive termite mounds represent.

Simple Summary: Termites are social insects that invade plantations, agricultural crops, and man-made structures throughout the world. The districts Buner, Haripur, and Swabi belong to different agro-ecological zones and a neglected area of Khyber Pakhtunkhwa (Pakistan: Oriental region) for work on the distribution and identity of termites. Termites were collected and identified morphologically and with DNA barcoding of the COII region. Odontotermes assmuthi , Odontotermes obesus , Odontotermes parvidens , and Odontotermes horai were identified, and a key to the genus Odontotermes of the area was made along with a distribution map. The identified species were found feeding on different forage substrates. Four novel COII sequences were submitted to GenBank. Abstract: The neglected area of Khyber Pakhtunkhwa (Pakistan: Oriental region), consisting of Buner, Haripur, and Swabi districts, were surveyed for termites during the summer of 2016–2019 for identification and assessment of the distribution of colonies. Collections were made either directly from visible galleries or using traps with ethanol. Soldiers were used for morphometric identification and DNA extraction. Morphometric identification was carried out based on the available literature through measurements of 20 characters/indices and evaluating species differences statistically. Based on these characteristics, we generated a key and a distribution map of the genus Odontotermes for the study area. This is the first record of Odontotermes assmuthi and Odontotermes obesus in these three districts, the first record of Odontotermes parvidens for the Buner and Swabi districts, and the first record of Odontotermes horai for Haripur. We subsequently used barcoding of the mtDNA COII to verify species assignments of colonies and for phylogenetic analyses using Neighbor-Joining and Maximum Likelihood analyses.

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth's ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis.IMPORTANCEActinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis.

14 Bioconversion of hemicelluloses into simpler sugars leads to production of a significant amount of 15 pentose sugars, such as D-xylose. However, efficient utilization of pentoses by conventional yeast 16 production strains remains challenging. Wild yeast strains can provide new industrially relevant 17 characteristics and efficiently utilize pentose sugars. To explore this strategy, we isolated gut18 residing yeasts from the termite Macrotermes bellicosus collected in Comoé National Park, Côte 19 d ́Ivoire. The yeasts were classified through their ITS/LSU sequence, their genomes were 20 sequenced and annotated. We identified a novel yeast species, which we name Barnettozyma 21 botsteinii sp. nov. 1118 T (MycoBank: 833563, CBS 16679 T and IBT 710) and two new strains of 22 Kurtzmaniella quercitrusa: var. comoensis (CBS 16678, IBT 709) and var. filamentosus (CBS 23 16680, IBT 711). The two K. quercitrusa strains grow 15% faster on synthetic glucose medium 24 than Saccharomyces cerevisiae CEN.PK T in acidic conditions (pH = 3.2) and both strains grow on 25 D-xylose as the sole carbon source at a rate of 0.35 h -1 . At neutral pH, the yeast form of K. 26 quercitrusa var. filamentosus, but not var. comoensis, switched to filamentous growth in a carbon 27 source dependent manner. Their genomes are 11.0-13.2 Mb in size and contain between 4888 and 28 5475 predicted genes. Together with closely related species, we did not find any relationship 29 between gene content and ability to grow on xylose. Besides its metabolism, K. quercitrusa var. 30 filamentosus also has a large potential as a production organism, because of its capacity to grow at 31 low pH and to undergo a dimorphic shift. 32 D ow naded rom http/academ ic.p.com /g3journal/advance-a.1093/g3journal/jkab342/6377786 by Fculty of ife Scnces Lrary user on 04 O cber 2021

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.

Escherichia coli is a clinically important bacterial species implicated in human- and livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals.